The invention relates to the identification and characterization of microorganisms, particularly of bacteria, on the basis of a mass spectrometric measurement of their protein profiles with ionization by matrix-assisted laser desorption. Many types of microorganism (also termed microbes below), especially bacteria and unicellular fungi, can be very easily identified mass spectrometrically using a recently discovered method in which small quantities of microbes from a colony cultivated in the usual way on a nutrient medium are transferred to a mass spectrometric sample support plate, where they are measured directly with a mass spectrometer. The mass spectrum particularly represents the proteins of different masses if they are present in sufficient concentration in the microbes. Spectral libraries with hundreds or thousands of microbe spectra are then used to establish the identity of the microbes from this microbial protein profile.
The nutrient medium is usually in a moist gelatin in a Petri dish, thus making it possible to cultivate strains, each of which is pure, in separate microbe colonies simply and in the completely normal way. A quantity of microbes is transferred with a small spatula from a selected colony onto the mass spectrometric sample support, where the microbes are sprinkled with a solution of a conventional matrix substance for ionization by matrix-assisted laser desorption (MALDI). The organic solvent penetrates into the microbial cells and destroys them. The sample is then dried by evaporating the solvent and hence the dissolved matrix material crystallizes. Soluble proteins and peptides and, to a lesser extent, other substances of the cell are thus embedded into the matrix crystals.
The matrix crystals with the embedded analyte molecules are then bombarded with flashes of laser light in a mass spectrometer, thus creating ions of the analyte molecules which can then be measured separately according to the mass of the ions in the mass spectrometer. It is preferable to use time-of-flight mass spectrometers for this purpose. The mass spectrum is the profile of the mass values of these peptide ions, protein ions and other analyte ions. This profile is very characteristic of the types of microbe in question because each type of microbe produces its own, genetically predetermined proteins, each having characteristic masses. The protein profiles are characteristic of the microbes in the same way that fingerprints are characteristic of humans. Many laboratories, including central, state-controlled institutions for disease monitoring and prevention, are working on reliable and legally applicable (so-called “validated”) mass spectrometric libraries of protein profiles of microbes.
This simple method can even be used to distinguish between closely related sub-strains of microbes since the proteins of the microbes are genetically predefined and vary clearly in the sub-strains. Slight changes in the genetic blueprint create proteins with a necessarily different structure, whose masses are different to those of proteins whose structure has not been genetically modified; they thus have a different protein profile. New taxonomic classifications of microbes can even be performed in this way.
The masses of the proteins of completely identical types of microbe are naturally always the same and thus strictly reproducible; the intensities of the protein signals, on the other hand, can only be reproduced approximately. The use of different nutrient media for the cultivation affects the metabolism of the microbes and hence affects the quantities of the different proteins which are produced and their intensity in the protein profile. The effect is not great, however. The intensity fluctuations do not interfere with the identification if the computer programs are adapted accordingly. Likewise, the degree of maturity of the colonies only affects the intensities of the protein signals with respect to each other in the mass spectra, but here, as well, the effect is only slight. Mass spectra which have different characteristics for the same type of microbe only really exist in the case of spore-forming organisms: the spores have protein profiles which are different to those of normal cells.
The computer programs for searching libraries by comparing spectra take account of intensity fluctuations; the intensities play only a minor role here. Current analyses have shown that the identification of the microbes with these programs appears to be very reliable. The programs operate only via the similarity of the mass spectra, without individually identifying the proteins involved; the masses are rigorously incorporated into the search for similarities, the intensities much less rigorously. In particular, it is even possible for several proteins to be missing from the mass spectra (very low intensity) without this interfering with the similarity determination: It suffices for the identification that the mass values for the majority of proteins match. The library spectra are also able to store information as to which protein signals definitely have to be present, for example by storing threshold values for the intensities.
The above, briefly described method of using a small spatula to spread some microbes from a colony onto a reserved spot of a mass spectrometric sample support, which is then sprinkled with a matrix solution, is the simplest and, as yet, fastest type of sample preparation. The method can also be automated with the help of image recognizing pipetting robots for use in routine laboratories. After cultivating a colony which is only just visible, it takes only one or two hours until the identification is complete even if hundreds of samples are analyzed at the same time. Mass spectrometric sample supports each holding 384 samples are commercially available; scanning these mass spectra takes around one to two hours. If the task is urgent, individual microbe samples can be identified in a few minutes.
Other methods of sample preparation such as extracting the proteins after ultrasonic destruction (sonication) of the microbes, or centrifugal extraction have also been investigated. These methods provide spectra which are without exception surprisingly similar.
Nowadays, the mass spectra of the microbe proteins are scanned in linear time-of-flight mass spectrometers because these have a particularly high detection sensitivity, although the mass resolution of the spectra and the accuracy of the masses are considerably better from time-of-flight mass spectrometers with reflectors. In reflector mode, only around a twentieth of the ion signals appear, however, and the detection sensitivity is one to two orders of magnitude worse. The high sensitivity of the linear mode of a time-of-flight mass spectrometer is due to the fact that not only the stable ions are detected but also the fragment ions from so-called “metastable” decays of the ions. Even the neutral particles which are created en route from ion decays are measured. All these fragment ions and neutral particles, which have originated from one species of parent ion, have the same speed as the parent ions and thus arrive at the ion detector at the same time. The arrival time is a measure of the mass of the originally undecayed ions.
The increased detection sensitivity is so crucial for many applications that many of the disadvantages of the linear mode of operation of the time-of-flight mass spectrometer are tolerated. The energy of the desorbing and ionizing laser is increased for these applications, something which increases the ion yield but also increases their instability, although this does not matter here.
Acquiring mass spectra with time-of-flight mass spectrometers generally requires that a very high number of individual spectra are measured in rapid succession, said individual spectra usually being added together measurement point by measurement point to form a sum spectrum. The ions for each individual spectrum are generated by one laser bombardment for each spectrum. The sum spectra have to be generated in this way because of the low dynamic range of measurement in the individual spectrum. A minimum of approx. 50, in some cases even 1,000 or more individual spectra are measured here; in general, a sum spectrum consists of several hundred individual spectra, which modern mass spectrometers measure and add together in a few seconds. The total duration of a sum spectrum acquisition depends on the number of individual spectra and the bombardment frequency of the laser used. Lasers with 20 to 200 hertz are now used for this purpose; it takes around two to 20 seconds to acquire a good sum spectrum.
In the above-described fields of application, mass spectra are measured which reach into high mass ranges of 20,000 Daltons, for example. As previously mentioned, the low mass resolution means that in most parts of the mass spectrum it is no longer possible to resolve the isotope groups, which consist of ion signals which each differ by one Dalton. It is thus only the envelopes of the isotope groups which are measured. Mass spectrometric measuring methods have also become known which provide a higher resolution and a higher mass accuracy; it is not yet known, however, if they achieve comparable sensitivities.
This method of quickly and simply identifying microbes can be used in many areas, for example for monitoring drinking water or quality control in food production. In the case of food production, it is the type of microorganisms present which determines if the food is safe to consume. Suffice it here to mention harmful staphylococci, streptococci and salmonellae, which have to be found by continuous controls. On the other hand, beer, wine, cheese and yoghurt cannot be produced without the beneficial deployment of billions of microbes. The crucial thing here is that the strains are pure.
Strict monitoring is also necessary in the medical field. The main thing is to keep infective pathogens away from hospitals. Constant monitoring and identification of the ubiquitous microbes is a mandatory legal requirement for operating rooms, for example.
The identification of microbes is particularly important with infectious diseases. It is important that the type of pathogen can be identified very quickly in order that the correct medical care can be provided immediately. Mass spectrometric identification has also proven successful here although it has not yet become established.
In the medical field, there is not only the problem of fast identification, but also the problem of identifying resistances to the commonly used antibiotics. It is not possible to fight the disease quickly if the resistances are unknown. What is therefore required is not only fast identification but also fast determination and characterization of the resistances of microorganisms.
Until now, the determination of the resistances has been predominantly based on cultivation experiments of the microorganisms in different nutrient media containing bactericides or other antibiotics. These experiments are protracted and labor-intensive. They take at least 24 hours, usually even two days. Experiments are currently being carried out to identify the resistances using analyses of DNA sequences in the plasmids of the bacteria. The resistances are coded in the plasmids. This type of analytical method is very promising, but has not gained acceptance as yet.
The problem of microorganisms being resistant to antibiotics such as bactericides or fungicides is becoming more and more critical as time goes on. On the one hand, the speed with which microorganisms form resistance to different types of antibiotics is increasing; on the other hand, fewer and fewer new antibiotics with a medical application are being developed. It is now known that the resistances are not formed by new types of mutation and their selection, but by the interchange of plasmids between microorganisms, even between different types of microorganism. The microorganisms infect each other with the resistances.
There are many reasons for the rapid increase in resistances: irresponsible prescribing of antibiotics even when they are not necessary; courses of treatment with bactericides which are rashly broken off before the infective agents have been completely eradicated; irresponsible use in agriculture and livestock farming, often as a purely preventative measure. All these types of behavior help the selection of resistant types of microbes over the non-resistant types.
On the other hand, fewer and fewer new antibiotics are being developed. Since many new antibiotics have to be taken off the market after only a short time because they became ineffective, it is becoming less and less viable for the pharmaceutical companies to invest large amounts of money in developing antibiotics when this is becoming more and more difficult. According to the WHO, only three new antibiotics have come onto the market since 1990, whereas there were ten between 1940 and 1950 and five between 1971 and 1980.
Since the discovery of antibiotics by Alexander Fleming in 1929 (penicillin), well over 2,000 different types of antibiotic substance have been discovered, although the toxicity and side effects of many of them mean that only around 30 are used widely as chemotherapeutic drugs. The most effective antibiotics are frequently artificially produced derivatives of natural vegetable (from fungi and algae) or animal antibiotics; completely synthetic antibiotics are also available. Antibiotics act in very different ways: Some antibiotics positively destroy the microbes; others lead to the “quiet” death of the cell whereas others are only growth inhibitors and leave the combating and destruction of the inhibited, and hence weakened, microbes to the healing immune system of the patient concerned, whether human, animal or plant.
In the middle of the last century, antibiotics were acclaimed as the great hope for fighting infectious diseases, whereas they are now threatening to quickly become a blunt instrument. The only hope of salvation is through targeted use with courses of treatment which are completed in full, and this requires rapid identification of the infective pathogens and also the fast identification of their specific resistances to various types of antibiotics.